The present application is concerned with reagents and methods for the formation of disulfide bonds and/or for the chemical modification of proteins, in particular reagents and methods for use in the glycosylation of proteins.
The co- and post-translational glycosylation of proteins plays a vital role in their biological behaviour and stability (R. Dwek, Chem. Rev., 96:683-720 (1996)). For example, glycosylation plays a major role in essential biological processes such as cell signalling and regulation, development and immunity. The study of these events is made difficult by the fact that glycoproteins occur naturally as mixtures of so-called glycoforms that possess the same peptide backbone but differ in both the nature and the site of glycosylation. Furthermore, since protein glycosylation is not under direct genetic control, the expression of therapeutic glycoproteins in mammalian cell culture leads to heterogeneous mixtures of glycoforms. The ability to synthesise homogeneous glycoprotein glycoforms is therefore not only a prerequisite for accurate investigation purposes, but is of increasing importance when preparing therapeutic glycoproteins, which are currently marketed as multi-glycoform mixtures (e.g. erythropoietin and interleukins). Other post translational modifications of proteins, such as phosphorylation and methylation, are also of importance. Controlling the degree and nature of such modification of a protein therefore allows the possibility of investigating and controlling its behaviour in biological systems (B. G. Davis, Science, Vol 303, p 480-482, 2004).
A number of methods for the glycosylation of proteins are known, including chemical synthesis. Chemical synthesis of glycoproteins offers certain advantages, not least the possibility of access to pure glycoprotein glycoforms. One known synthetic method utilises thiol-selective carbohydrate reagents, glycosylmethane thiosulfonate reagents (glyco-MTS). Such glycosylmethane thiosulfonate reagents react with thiol groups in a protein to introduce a glycosyl residue linked to the protein via a disulfide bond (see for example WO00/01712).
However, glyco-MTS reagents suffer from a number of disadvantages, including occasionally moderate reaction yields, difficulties in their preparation and problems with stability under the basic conditions in which they are often used. There is therefore a need for further reagents for use in protein glycosylation which are readily prepared, stable and give high yields of the glycosylated protein product.
There is also a need for alternative methods for protein glycosylation which give high yields of the glycosylated protein product, are site-selective, and which allow glycosylation at both single and multiple sites in a wide range of different proteins.